Glucoamylase variants and polynucleotides encoding same

ABSTRACT

The present invention relates to glucoamylase variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to glucoamylase variants, polynucleotidesencoding the variants, methods of producing the variants, and methods ofusing the variants. Also described are the use of glucoamylases of theinvention for starch conversion to produce fermentation products, suchas ethanol, and syrups, such as glucose. The invention also relates to acomposition comprising a glucoamylase of the invention.

2. Description of the Related Art

Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) is anenzyme, which catalyzes the release of D-glucose from the non-reducingends of starch or related oligo- and polysaccharide molecules.Glucoamylases are produced by several filamentous fungi and yeast, withthose from Aspergillus being commercially most important.

Commercially, glucoamylases are used to convert starch containingmaterial, which is already partially hydrolyzed by an alpha-amylase, toglucose. The glucose may then be converted directly or indirectly into afermentation product using a fermenting organism. Examples of commercialfermentation products include alcohols (e.g., ethanol, methanol,butanol, 1,3-propanediol); organic acids (e.g., citric acid, aceticacid, itaconic acid, lactic acid, gluconic acid, gluconate, lactic acid,succinic acid, 2,5-diketo-D-gluconic acid); ketones (e.g., acetone);amino acids (e.g., glutamic acid); gases (e.g., H₂ and CO₂), and morecomplex compounds, including, for example, antibiotics (e.g., penicillinand tetracycline); enzymes; vitamins (e.g., riboflavin, B₁₂,beta-carotene); hormones, and other compounds which are difficult toproduce synthetically. Fermentation processes are also commonly used inthe consumable alcohol (e.g., beer and wine), dairy (e.g., in theproduction of yogurt and cheese) industries.

The end product may also be a syrup. For instance, the end product maybe glucose, but may also be converted, e.g., by a glucose isomerase tofructose or a mixture composed almost equally of glucose and fructose.This mixture, or a mixture further enriched with fructose, is the mostcommonly used high fructose corn syrup (HFCS) commercialized throughoutthe world.

It is an object of the present invention to provide polypeptides havingglucoamylase activity and polynucleotides encoding the polypeptides andwhich provide a high yield in fermentation product production processes,such as ethanol production processes, including one-step ethanolfermentation processes from un-gelatinized raw (or uncooked) starch.

WO2006/069289 discloses glucoamylases derived from Trametes cingulata,Pachykytospora papyracea and Leucopaxillus giganteus and the use thereofin processes for manufacturing fermentation products.

The present invention provides glucoamylase variants with improvedproperties compared to its parent.

SUMMARY OF THE INVENTION

The present invention relates to glucoamylase variant comprising asubstitution at one or more positions corresponding to positions 45, 46,61, 86, 119, 120, 318 and 348 of the polypeptide of SEQ ID NO: 3,wherein the variant has glucoamylase activity and wherein the varianthas at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99%, but lessthan 100% sequence identity to the polypeptide of SEQ ID NO: 3.

The present invention also relates to isolated polynucleotides encodingthe variants; nucleic acid constructs, vectors, and host cellscomprising the polynucleotides; and methods of producing the variants.

The present invention also relates to compositions comprising theglucoamylase variant of the invention.

The present invention further relates to a use of the glucoamylasevariant of the invention for producing a syrup or a fermentationproduct.

The present invention further relates to a process of producing afermentation product from starch-containing material comprising thesteps of: (a) liquefying starch-containing material in the presence ofan alpha amylase; (b) saccharifying the liquefied material; and (c)fermenting with a fermenting organism; wherein step (a) and/or step (b)is carried out using at least a glucoamylase variant.

The present invention still further relates to a process of producing afermentation product from starch-containing material, comprising thesteps of: (a) saccharifying starch-containing material at a temperaturebelow the initial gelatinization temperature of said starch-containingmaterial; and (b) fermenting with a fermenting organism, wherein step(a) is carried out using at least a glucoamylase variant.

DEFINITIONS

Glucoamylase: The term “glucoamylase” (1,4-alpha-D-glucanglucohydrolase, EC 3.2.1.3) is defined as an enzyme, which catalyzes therelease of D-glucose from the non-reducing ends of starch or relatedoligo- and polysaccharide molecules. For purposes of the presentinvention, glucoamylase activity is determined according to theprocedure described in the Examples herein. The Glucoamylase Unit (AGU)is defined as the amount of enzyme, which hydrolyses 1 micromole maltoseper minute under the standard conditions 37° C., pH 4.3, substrate:maltose 23.2 mM, buffer: acetate 0.1 M, reaction time 5 minutes.

The polypeptides of the present invention have at least 20%, preferablyat least 40%, preferably at least 45%, more preferably at least 50%,more preferably at least 55%, more preferably at least 60%, morepreferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 100% of the glucoamylase activity ofthe mature polypeptide of SEQ ID NO: 2.

In another embodiment the polypeptides of the present invention have atleast 20%, preferably at least 40%, preferably at least 45%, morepreferably at least 50%, more preferably at least 55%, more preferablyat least 60%, more preferably at least 65%, more preferably at least70%, more preferably at least 75%, more preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 100% of theglucoamylase activity of the polypeptide of SEQ ID NO: 3.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a variant. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding avariant of the present invention. Each control sequence may be native(i.e., from the same gene) or foreign (i.e., from a different gene) tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Expression: The term “expression” includes any step involved in theproduction of a variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to control sequences that provide for itsexpression.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment has glucoamylaseactivity. In one aspect, a fragment contains at least 450 amino acidresidues (e.g., amino acids 23 to 472 of SEQ ID NO: 2) comprising thecatalytic domain and having one or more of the substitutions accordingto the invention.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Improved property: The term “improved property” means a characteristicassociated with a variant that is improved compared to the parent. Suchan improved property is thermostability.

Isolated: The term “isolated” means a substance in a form or environmentwhich does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 19 to 574 of SEQ ID NO: 2 and amino acids 1to 18 of SEQ ID NO: 2 are a signal peptide. It is known in the art thata host cell may produce a mixture of two of more different maturepolypeptides (i.e., with a different C-terminal and/or N-terminal aminoacid) expressed by the same polynucleotide. The mature polypeptide isdisclosed herein as SEQ ID NO: 3.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving glucoamylase activity. In one aspect, the mature polypeptidecoding sequence is nucleotides 55 to 1722 of SEQ ID NO: 1 andnucleotides 1 to 54 of SEQ ID NO: 1 encode a signal peptide.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 60° C.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Parent or parent [enzyme]: The term “parent” or “parent [glucoamylase]”means a glucoamylase to which an alteration is made to produce theenzyme variants of the present invention. The parent may be a naturallyoccurring (wild-type) polypeptide or a variant or fragment thereof.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the -nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having glucoamylase activity. In one aspect, a subsequencecomprises the catalytic domain and contains at least 1350 nucleotides(e.g., nucleotides 67 to 1416 of SEQ ID NO: 1).

Variant: The term “variant” means a polypeptide having glucoamylaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition

The variants of the present invention have at least 20%, e.g., at least40%, at least 45%, more preferably at least 50%, more preferably atleast 55%, more preferably at least 60%, more preferably at least 65%,more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 100% of the glucoamylase activity of the maturepolypeptide of SEQ ID NO: 2 disclosed as SEQ ID NO: 3.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

Wild-type glucoamylase: The term “wild-type” glucoamylase means aglucoamylase expressed by a naturally occurring microorganism, such as abacterium, yeast, or filamentous fungus found in nature.

Conventions for Designation of Variants

For purposes of the present invention, the mature polypeptide disclosedin SEQ ID NO: 3 is used to determine the corresponding amino acidresidue in another glucoamylase. The amino acid sequence of anotherglucoamylase is aligned with the mature polypeptide disclosed in SEQ IDNO: 3, and based on the alignment, the amino acid position numbercorresponding to any amino acid residue in the mature polypeptidedisclosed in SEQ ID NO: 3 is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends Genet. 16: 276-277), preferably version 5.0.0 or later. Theparameters used are gap open penalty of 10, gap extension penalty of0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

Identification of the corresponding amino acid residue in anotherglucoamylase can be determined by an alignment of multiple polypeptidesequences using several computer programs including, but not limited to,MUSCLE (multiple sequence comparison by log-expectation; version 3.5 orlater; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT(version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518;Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009,Methods in Molecular Biology 537:_39-64; Katoh and Toh, 2010,Bioinformatics 26:_1899-1900), and EMBOSS EMMA employing ClustalW (1.83or later; Thompson et al., 1194, Nucleic Acids Research 22: 4673-4680),using their respective default parameters.

When the other enzyme has diverged from the mature polypeptide of SEQ IDNO: 2 such that traditional sequence-based comparison fails to detecttheir relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), other pairwise sequence comparison algorithms can be used.Greater sensitivity in sequence-based searching can be attained usingsearch programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1197, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones, 1199, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1198, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1198,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviation is employed.

Substitutions.

For an amino acid substitution, the following nomenclature is used:Original amino acid, position, substituted amino acid. Accordingly, thesubstitution of threonine at position 226 with alanine is designated as“Thr226Ala” or “T226A”. Multiple mutations are separated by additionmarks (“+”), e.g., “Gly205Arg+Ser411Phe” or “G205R+5411 F”, representingsubstitutions at positions 205 and 411 of glycine (G) with arginine (R)and serine (S) with phenylalanine (F), respectively.

Deletions.

For an amino acid deletion, the following nomenclature is used: Originalamino acid, position, *. Accordingly, the deletion of glycine atposition 195 is designated as “Gly195*” or “G195*”. Multiple deletionsare separated by addition marks (“+”), e.g., “Gly195*+Ser411*” or“G195*+S411*”.

Insertions.

For an amino acid insertion, the following nomenclature is used:Original amino acid, position, original amino acid, inserted amino acid.Accordingly the insertion of lysine after glycine at position 195 isdesignated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G—K—A

Multiple Alterations.

Variants comprising multiple alterations are separated by addition marks(“+”), e.g., “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing asubstitution of arginine and glycine at positions 170 and 195 withtyrosine and glutamic acid, respectively.

Different Alterations.

Where different alterations can be introduced at a position, thedifferent alterations are separated by a comma, e.g., “Arg170Tyr,Glu”represents a substitution of arginine at position 170 with tyrosine orglutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala” designates thefollowing variants:

“Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and“Tyr167Ala+Arg170Ala”. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to glucoamylase variants, comprising ansubstitution at one or more positions corresponding to positions 45, 46,61, 86, 119, 120, 318 and 348 of the polypeptide of SEQ ID NO: 3,wherein the variant has glucoamylase activity. In particular thevariants have improved property compared to the glucoamylase disclosedas SEQ ID NO: 3. Particularly, the improved property is increasedthermo-stability. The variants according to the invention have at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%sequence identity to the polypeptide of SEQ ID NO: 3.

The mature polypeptide of SEQ ID NO: 2 corresponds to SEQ ID NO: 3. Thusthe position numbers referred to herein correspond to the positionnumbers of SEQ ID NO: 3.

Variants

The present invention provides glucoamylase variants, comprising ansubstitution at one or more (e.g., several) positions corresponding topositions 45, 46, 61, 86, 119, 120, 318 and 348 and the variant hasglucoamylase activity.

In one embodiment, the variant is isolated.

In another embodiment, the variant has sequence identity of at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%, but less than 100%, to the aminoacid sequence of the parent glucoamylase.

In yet another embodiment, the variant has at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, such as at least 96%, at least97%, at least 98%, or at least 99%, but less than 100%, sequenceidentity to the mature polypeptide of SEQ ID NO: 2. The maturepolypeptide of SEQ ID NO: 2 is in one embodiment of SEQ ID NO: 3.

In a further embodiment, the present invention relates to a glucoamylasevariant comprising a substitution at one or more positions correspondingto positions 45, 46, 61, 86, 119, 120, 318 and 348 of the polypeptide ofSEQ ID NO: 3, wherein the variant has glucoamylase activity and whereinthe variant has at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 3.In one aspect, the number of susbtitutions in the variants of thepresent invention is 1-20, e.g., 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 alterations.

In another aspect, a variant comprises an subsitution at one or morepositions corresponding to positions 45, 46, 61, 86, 119, 120, 318 and348. In another aspect, a variant comprises an substitution at twopositions corresponding to any of positions 45, 46, 61, 86, 119, 120,318 and 348. In another aspect, a variant comprises an substitution atthree positions corresponding to any of positions 45, 46, 61, 86, 119,120, 318 and 348. In another aspect, a variant comprises an substitutionat four positions corresponding to any of positions 45, 46, 61, 86, 119,120, 318 and 348. In another aspect, a variant comprises an substitutionat five positions corresponding to any of positions 45, 46, 61, 86, 119,120, 318 and 348. In another aspect, a variant comprises an substitutionat six positions corresponding to any of positions 45, 46, 61, 86, 119,120, 318 and 348. In another aspect, a variant comprises an subsitutionat each position corresponding to positions 45, 46, 61, 86, 119, 120,318 and 348.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 45. In another aspect, the aminoacid at a position corresponding to position 45 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Trp, Tyr, or Val, preferably with Lys. In another aspect, thevariant comprise or consist of the substitution T45K of the maturepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 46. In another aspect, the aminoacid at a position corresponding to position 46 is substituted with Ala,Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Thr, Trp, Tyr, or Val, preferably with His. In another aspect, thevariant comprise or consist of the substitution S46H of the maturepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 61. In another aspect, the aminoacid at a position corresponding to position 61 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp or Tyr, preferably with Thr or Ala. In another aspect, thevariant comprise or consist of the substitution V61T or V61A of themature polypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 86. In another aspect, the aminoacid at a position corresponding to position 86 is substituted with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Thr, Trp, Tyr, or Val, preferably with Cys. In another aspect, thevariant comprise or consist of the substitution S86C of the maturepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 119. In another aspect, theamino acid at a position corresponding to position 119 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Trp, Tyr, or Val, preferably with Trp. In another aspect,the variant comprise or consist of the substitution T119W of the maturepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 120. In another aspect, theamino acid at a position corresponding to position 120 is substitutedwith Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Gly. In another aspect,the variant comprise or consist of the substitution D120G of the maturepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 318. In another aspect, theamino acid at a position corresponding to position 318 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Trp. In another aspect,the variant comprise or consist of the substitution M318W of the maturepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 348. In another aspect, theamino acid at a position corresponding to position 348 is substitutedwith Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Thr. In another aspect,the variant comprise or consist of the substitution N348T of the maturepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 46, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 61, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 86, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 119, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 120, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 318, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 348, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 46 and 61, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 46 and 86, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 46 and 119, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 46 and 120, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 46 and 318, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 46 and 348, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 61 and 86, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 61 and 119, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 61 and 120, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 61 and 318, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 61 and 348, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 86 and 119, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 86 and 120, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 86 and 318, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 86 and 348, such as those describedabove.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 119 and 120, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 119 and 318, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 119 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 120 and 318, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 120 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 318 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 46 and 61, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 46 and 86, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 46 and 119, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 46 and 120, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 46 and 318, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 46 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 61 and 86, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 61 and 119, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 61 and 120, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 61 and 318, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 61 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 86 and 119, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 86 and 120, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 86 and 318, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 86 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 119 and 120, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 119 and 318, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 119 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 120 and 318, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 120 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 45 and 318 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 46 and 61 and 86, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 46 and 61 and 119, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 46 and 61 and 120, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 46 and 61 and 318, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 46 and 61 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 46 and 61 and 119, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 46 and 61 and 120, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 46 and 86 and 318, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 46 and 86 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 46 and 119 and 120, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 46 and 119 and 318, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 46 and 119 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 46 and 120 and 318, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 46 and 120 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 46 and 318 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 61 and 86 and 119, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 61 and 86 and 120, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 61 and 86 and 318, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 61 and 86 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 61 and 119 and 120, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 61 and 119 and 318, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 61 and 119 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 61 and 120 and 318, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 61 and 120 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 61 and 318 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 86 and 119 and 120, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 86 and 119 and 318, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 86 and 119 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 86 and 120 and 318, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 86 and 120 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 86 and 318 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 119 and 120 and 318, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 119 and 120 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 119 and 318 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 120 and 318 and 348, such as thosedescribed above.

In another aspect, the variant comprises or consists of one or moresubstitutions selected from the group consisting of T45K; S46H; V61T;S86C; T119W; D120G; M318W and N348T and wherein the variant hasglucoamylase activity and wherein the variant has at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%, but less than 100% sequence identityto the polypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of the below listedspecific substitutions or combinations of specific substutions of themature polypeptide of SEQ ID NO: 3.

T119W; D120G; V61A; S86C+T119W; T45K+T119W; T119W+D120G; T45K+D120G;V61T+T119W; D120G+M318W; T45K+S46H; T119W+D120G+N348T;T119W+D120G+M318W; S86C+T119W+D120G; S46H+T119W+D120G;

T45K+T119W+D120G and wherein the variant has glucoamylase activity andwherein the variant has at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment toglucoamylase variants, comprising the substitution T119W of thepolypeptide of SEQ ID NO: 3, wherein the variant has glucoamylaseactivity, and wherein the glucoamylase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment toglucoamylase variants, comprising the substitution D120G of thepolypeptide of SEQ ID NO: 3, wherein the variant has glucoamylaseactivity, and wherein the glucoamylase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment toglucoamylase variants, comprising the substitution S86C+T119W of thepolypeptide of SEQ ID NO: 3, wherein the variant has glucoamylaseactivity, and wherein the glucoamylase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment toglucoamylase variants, comprising the substitution T45K+T119W of thepolypeptide of SEQ ID NO: 3, wherein the variant has glucoamylaseactivity, and wherein the glucoamylase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment toglucoamylase variants, comprising the substitution T119W+D120G of thepolypeptide of SEQ ID NO: 3, wherein the variant has glucoamylaseactivity, and wherein the glucoamylase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment toglucoamylase variants, comprising the substitution T45K+D120G of thepolypeptide of SEQ ID NO: 3, wherein the variant has glucoamylaseactivity, and wherein the glucoamylase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment toglucoamylase variants, comprising the substitution V61T+T119W of thepolypeptide of SEQ ID NO: 3, wherein the variant has glucoamylaseactivity, and wherein the glucoamylase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment toglucoamylase variants, comprising the substitution D120G+M318W of thepolypeptide of SEQ ID NO: 3, wherein the variant has glucoamylaseactivity, and wherein the glucoamylase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment toglucoamylase variants, comprising the substitution T45K+S46H of thepolypeptide of SEQ ID NO: 3, wherein the variant has glucoamylaseactivity, and wherein the glucoamylase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment toglucoamylase variants, comprising the substitution T119W+D120G+N348T ofthe polypeptide of SEQ ID NO: 3, wherein the variant has glucoamylaseactivity, and wherein the glucoamylase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment toglucoamylase variants, comprising the substitution T119W+D120G+M318W ofthe polypeptide of SEQ ID NO: 3, wherein the variant has glucoamylaseactivity, and wherein the glucoamylase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment toglucoamylase variants, comprising the substitution S86C+T119W+D120G ofthe polypeptide of SEQ ID NO: 3, wherein the variant has glucoamylaseactivity, and wherein the glucoamylase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment toglucoamylase variants, comprising the substitution S46H+T119W+D120G ofthe polypeptide of SEQ ID NO: 3, wherein the variant has glucoamylaseactivity, and wherein the glucoamylase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment toglucoamylase variants, comprising the substitution T45K+T119W+D120G ofthe polypeptide of SEQ ID NO: 3, wherein the variant has glucoamylaseactivity, and wherein the glucoamylase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

All of the above specific substitutions or combinations of substitutionshave an effect of increasing the thermo-stability of the resultingvariant when introduced in the parent disclosed as SEQ ID NO: 3.

The variants may further comprise one or more additional substitutionsat one or more (e.g., several) other positions. Such further variationcould be introduced without affecting significantly the properties ofthe glucoamylase variants.

Therefore the % identity of the variant polypeptide compared to theparent polypeptide of SEQ ID NO: 3 may be at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, such as at least 96%, at least 97%, at least98%, or at least 99%, but less than 100%, sequence identity to thepolypeptide of SEQ ID NO: 3.

In one particular embodiment the above variants have glucoamylaseactivity, and the variant has at least 75%, but less than 100%, sequenceidentity to the polypeptide of SEQ ID NO: 3.

In one particular embodiment the above variants have glucoamylaseactivity, and the variant has at least 80%, but less than 100%, sequenceidentity to the polypeptide of SEQ ID NO: 3.

In one particular embodiment the above variants have glucoamylaseactivity, and the variant has at least 85%, but less than 100%, sequenceidentity to the polypeptide of SEQ ID NO: 3.

In one particular embodiment the above variants have glucoamylaseactivity, and the variant has at least 90%, but less than 100%, sequenceidentity to the polypeptide of SEQ ID NO: 3.

In one particular embodiment the above variants have glucoamylaseactivity, and the variant has at least 91%, but less than 100%, sequenceidentity to the polypeptide of SEQ ID NO: 3.

In one particular embodiment the above variants have glucoamylaseactivity, and the variant has at least 92%, but less than 100%, sequenceidentity to the polypeptide of SEQ ID NO: 3.

In one particular embodiment the above variants have glucoamylaseactivity, and the variant has at least 93%, but less than 100%, sequenceidentity to the polypeptide of SEQ ID NO: 3.

In one particular embodiment the above variants have glucoamylaseactivity, and the variant has at least 94%, but less than 100%, sequenceidentity to the polypeptide of SEQ ID NO: 3.

In one particular embodiment the above variants have glucoamylaseactivity, and the variant has at least 95%, but less than 100%, sequenceidentity to the polypeptide of SEQ ID NO: 3.

In one particular embodiment the above variants have glucoamylaseactivity, and the variant has at least 96%, but less than 100%, sequenceidentity to the polypeptide of SEQ ID NO: 3.

In one particular embodiment the above variants have glucoamylaseactivity, and the variant has at least 97%, but less than 100%, sequenceidentity to the polypeptide of SEQ ID NO: 3.

In one particular embodiment the above variants have glucoamylaseactivity, and the variant has at least 98%, but less than 100%, sequenceidentity to the polypeptide of SEQ ID NO: 3.

In one particular embodiment the above variants have glucoamylaseactivity, and the variant has at least 99%, but less than 100%, sequenceidentity to the polypeptide of SEQ ID NO: 3.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for glucoamylase activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1196, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1192, Science 255:306-312; Smith et al., 1192, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1192, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

In an embodiment, the variant has improved thermostability compared tothe parent enzyme. The improved thermostability may be determined bymeasuring the residual glucoamylase activity as disclosed in theexamples.

Parent Glucoamylase

The parent glucoamylase may be (a) a polypeptide having at least 70%sequence identity to the mature polypeptide of SEQ ID NO: 2; (b) apolypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, or (ii) the full-length complement of (i) or (c) apolypeptide encoded by a polynucleotide having at least 70% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1.

In an aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 2 of at least 75%, at least 80%, at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%, which have glucoamylase activity. In one aspect, the amino acidsequence of the parent differs by up to 10 amino acids, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 2.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 2. In another aspect, the parent comprises orconsists of the mature polypeptide of SEQ ID NO: 2. In another aspect,the parent comprises or consists of amino acids 18 to 574 of SEQ ID NO:2.

In another aspect, the parent is a fragment of the mature polypeptide ofSEQ ID NO: 2 containing at least 450 amino acid residues, e.g., at least23 and at least 472 amino acid residues.

In another embodiment, the parent is an allelic variant of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the parent is encoded by a polynucleotide thathybridizes under high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, or (ii) the full-length complement of (i) (Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well asthe polypeptide of SEQ ID NO: 2 or a fragment thereof, may be used todesign nucleic acid probes to identify and clone DNA encoding a parentfrom strains of different genera or species according to methods wellknown in the art. In particular, such probes can be used forhybridization with the genomic DNA or cDNA of a cell of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S. biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other strains may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith SEQ ID NO: 1 or a subsequence thereof, the carrier material is usedin a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQID NO: 1; (iiii) the full-length complement thereof; or (iv) asubsequence thereof; under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions can be detected using, for example, X-ray film or any otherdetection means known in the art.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1. In another aspect, the nucleic acid probe isnucleotides 55 to 1722 of SEQ ID NO: 1. In another aspect, the nucleicacid probe is a polynucleotide that encodes the polypeptide of SEQ IDNO: 2; the mature polypeptide thereof; or a fragment thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 1.

In another embodiment, the parent is encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptidein which another polypeptide is fused at the N-terminus or theC-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1193, EMBO J. 12:2575-2583; Dawson et al., 1194, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1197, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1195, Biotechnology 13:498-503; and Contreras et al., 1191, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1195,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The parent may be obtained from microorganisms of any genus. Forpurposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the parentencoded by a polynucleotide is produced by the source or by a strain inwhich the polynucleotide from the source has been inserted. In oneaspect, the parent is secreted extracellularly.

The parent may be a fungal glucoamylase. For example, the parent may bea Trametes glucoamylase.

In another aspect, the parent is a Trametes cingulate glucoamylase.

In another aspect, the parent is a Trametes cingulata glucoamylase e.g.,the glucoamylase of SEQ ID NO: 2 or the mature polypeptide thereof.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. A polynucleotide encoding a parent may then beobtained by similarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with the probe(s), the polynucleotide can beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1190, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1198, Nat. Med. 4: 285-290; and Calissano and Macino, 1196,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1191,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1199, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga variant of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the variant. The promoter may be any polynucleotide thatshows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1194,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1192, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the variant. Any terminator that is functional in the host cellmay be used.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1192, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1195, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leadersequence is operably linked to the 5′-terminus of the polynucleotideencoding the variant. Any leader that is functional in the host cell maybe used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the variant-encoding sequence and,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1195, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding sequence naturally linked in translation reading framewith the segment of the coding sequence that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1193, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1192, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a variant. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of the variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the variant would be operably linkedwith the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMR1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1191, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thevariant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see,e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1191, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1199, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see,e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any methodknown in the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1195, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1195, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a variant,comprising: (a) cultivating a host cell of the present invention underconditions suitable for expression of the variant; and (b) recoveringthe variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the variantto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the variants. These detection methods include, but are notlimited to, use of specific antibodies, formation of an enzyme product,or disappearance of an enzyme substrate. For example, an enzyme assaymay be used to determine the activity of the variant.

The variant may be recovered using methods known in the art. Forexample, the variant may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing the variant is used as asource of the variant.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably the composition alsocomprises a carrier and/or an excipient. More preferably, thecompositions are enriched in such a polypeptide. The term “enriched”indicates that the glucoamylase activity of the composition has beenincreased, e.g., with an enrichment factor of at least 1.1. Preferably,the compositions are formulated to provide desirable characteristicssuch as low color, low odor and acceptable storage stability.

The composition may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, alpha-amylase, isoamylasecarbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase,lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase,peroxidase, phytase, polyphenoloxidase, pullulanase, proteolytic enzyme,ribonuclease, transglutaminase, or xylanase.

In a particular embodiment the composition comprises an alpha-amylaseand glucoamylase variant according to the invention. In anotherembodiment the composition comprises an isoamylase and glucoamylasevariant according to the invention. In another embodiment thecomposition comprises an alpha-amylase, an isoamylase and theglucoamylase variant according to the invention.

In another aspect the composition comprises the glucoamylase variant ofthe invention combined with a pullulanase. In another aspect thecomposition comprises the glucoamylase variant of the invention combinedwith a pullulanase, and an isoamylase. In another aspect the compositioncomprises the glucoamylase variant of the invention combined with apullulanase, and an alpha-amylase.

In a particular embodiment the composition further comprises a protease.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art. Examples are given below of preferred uses of the polypeptideor polypeptide compositions of the invention. The dosage of thepolypeptide composition of the invention and other conditions underwhich the composition is used may be determined on the basis of methodsknown in the art.

The above compositions are suitable for use in liquefaction,saccharification, and/or fermentation processes, preferably in starchconversion, especially for producing syrup and fermentation products,such as ethanol.

Examples are given below of preferred uses of the polypeptidecompositions of the present invention. The dosage of the polypeptidecomposition of the invention and other conditions under which thecomposition is used may be determined on the basis of methods known inthe art.

Uses

The present invention is also directed to use of a polypeptide of thepresent invention in a liquefaction, a saccharification and/or afermentation process. The polypeptide may be used in a single process,for example, in a liquefaction process, a saccharification process, or afermentation process. The polypeptide may also be used in a combinationof processes for example in a liquefaction and saccharification process,in a liquefaction and fermentation process, or in a saccharification andfermentation process, preferably in relation to starch conversion.

In a preferred aspect of the present invention, the liquefaction,saccharification and/or fermentation process includes sequentially orsimultaneously performed liquefaction and saccharification processes.

In conventional enzymatic liquefaction process, thermostablealpha-amylase is added and the long chained starch is degraded intobranched and linear shorter units (maltodextrins), but glucoamylase isnot added. The glucoamylase of the present invention is highlythermostable, so it is advantageous to add the glucoamylase in theliquefaction process. The glucoamylase of the present invention has asynergistic effect when combined with an alpha-amylase in theliquefaction process. During conventional saccharification, the dextrinsgenerated during the liquefaction process are further hydrolyzed toproduce low molecular sugars DP1-3 that can be metabolized by fermentingorganism. The hydrolysis is typically accomplished using glucoamylases;alternatively in addition to glucoamylases, alpha-glucosidases and/oracid alpha-amylases can be used.

When applying the glucoamylase of the present invention, potentially incombination with an alpha-amylase in a liquefaction and/orsaccharification process, especially in a simultaneous liquefaction andsaccharification process, the process can be conducted at a highertemperature. In another aspect the composition comprises theglucoamylase variant of the invention combined with a pullulanase. Inanother aspect the composition comprises the glucoamylase variant of theinvention combined with a pullulanase, and an isoamylase. In anotheraspect the composition comprises the glucoamylase variant of theinvention combined with a pullulanase, and an alpha-amylase.

By conducting the liquefaction and/or saccharification process at highertemperatures the process can be carried out in a shorter period of timeor alternatively the process can be carried out using lower enzymedosage. Furthermore, the risk of microbial contamination is reduced whencarrying the liquefaction and/or saccharification process at highertemperature.

Conversion of Starch-Containing Material

The present invention provides a use of the glucoamylase of theinvention for producing glucoses and the like from starch. Generally,the method includes the steps of partially hydrolyzing precursor starchusing glucoamylase variant of the present invention either alone or inthe presence of an alpha-amylase.

The glucoamylase variant of the invention may also be used incombination with an enzyme that hydrolyzes only alpha-(1,6)-glucosidicbonds in molecules comprising at least four glucosyl residues.

In a further aspect the invention relates to the use of a glucoamylaseof the invention in starch conversion. Furthermore, the glucoamylase ofthe invention may be used in a continuous starch conversion processincluding a continuous saccharification process.

Production of Syrup, Beverage and/or Fermentation Product

Uses of the glucoamylase of the invention include conversion of starchto e.g., syrup beverage, and/or a fermentation product, includingethanol.

The present invention also provides a process of using a glucoamylase ofthe invention for producing syrup, such as glucose and the like, fromstarch-containing material. Suitable starting materials are exemplifiedin the “Starch-containing materials”-section. Generally, the processcomprises the steps of partially or totally hydrolyzingstarch-containing material (liquefaction and/or saccharification) in thepresence of the glucoamylase of the present invention alone or incombination with alpha-amylase to release glucose from the non-reducingends of the starch or related oligo- and poly-saccharide molecules.

The glucoamylase variant of the invention may also be used inimmobilized form. This is suitable and often used for producingspeciality syrups, such as maltose syrups as well as in the raffinatestream of oligosaccharides in connection with the production of fructosesyrups, e.g., high fructose syrup (HFS).

Fermentation Products

The term “fermentation product” means a product produced by a processincluding a fermentation process using a fermenting organism.Fermentation products contemplated according to the invention includealcohols (e.g., arabinitol, butanol, ethanol, glycerol, methanol,ethylene glycol, 1,3-propanediol [propylene glycol], butanediol,glycerin, sorbitol, and xylitol); organic acids (e.g., acetic acid,acetonic acid, adipic acid, ascorbic acid, citric acid,2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid,gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid,itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid,oxaloacetic acid, propionic acid, succinic acid, and xylonic acid);ketones (e.g., acetone); amino acids (e.g., aspartic acid, glutamicacid, glycine, lysine, serine, and threonine); an alkane (e.g., pentane,hexane, heptane, octane, nonane, decane, undecane, and dodecane); acycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, andcyclooctane); an alkene (e.g. pentene, hexene, heptene, and octene);gases (e.g., methane, hydrogen (H₂), carbon dioxide (CO₂), and carbonmonoxide (CO)); antibiotics (e.g., penicillin and tetracycline);enzymes; vitamins (e.g., riboflavin, B₁₂, beta-carotene); and hormones.In a preferred aspect the fermentation product is ethanol, e.g., fuelethanol; drinking ethanol, i.e., potable neutral spirits; or industrialethanol or products used in the consumable alcohol industry (e.g., beerand wine), dairy industry (e.g., fermented dairy products), leatherindustry and tobacco industry. Preferred beer types comprise ales,stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcoholbeer, low-alcohol beer, low-calorie beer or light beer. Preferredfermentation processes used include alcohol fermentation processes,which are well known in the art. Preferred fermentation processes areanaerobic fermentation processes, which are well known in the art.

Brewing

The glucoamylases of the present invention are highly thermostable andtherefore they can be used in an industry which needs starch hydrolysisat high temperature. For example, glucoamylases of the invention can beused in a brewing industry. The glucoamylases of the invention is addedin effective amounts which can be easily determined by the skilledperson in the art.

Production of a Liquefaction, Saccharification and/or FermentationProduct

In this aspect the present invention relates to a process for producinga liquefaction, saccharification and/or fermentation product fromstarch-containing material, comprising the step of: treatingstarch-containing material with a polypeptide of the present invention.

Suitable starch-containing starting materials are listed in the“Starch-containing materials”-section below. Contemplated enzymes arelisted in the “Enzymes”—section below. Preferably the process of presentinvention comprises treating starch-containing material with apolypeptide of the present invention alone or together with analpha-amylase. The liquefaction and/or saccharification product of thepresent invention are dextrin, or low molecular sugars, for exampleDP1-3. In the liquefaction process the conversion of starch intoglucose, dextrin and/or low molecular weight sugars is enhanced by theaddition of a glucoamylase of the present invention. The fermentationproduct, such as ethanol, may optionally be recovered afterfermentation, e.g., by distillation. The fermentation is preferablycarried out in the presence of yeast, preferably a strain ofSaccharomyces. Suitable fermenting organisms are listed in the“Fermenting Organisms”-section below.

Process for Producing Fermentation Products from Gelatinized StarchContaining Material

In this aspect the present invention relates to a process for producinga fermentation product, especially ethanol, from starch-containingmaterial, which process includes a liquefaction step and sequentially orsimultaneously performed saccharification and fermentation steps.

The invention relates to a process for producing a fermentation productfrom starch-containing material comprising the steps of:(a) liquefying starch-containing material; using an alpha amylase;(b) saccharifying the liquefied material obtained in step (a) using aglucoamylase; and(c) fermenting the saccharified material using a fermenting organism.Preferably step (a) includes also using the glucoamylase of theinvention. In one embodiment the glucoamylase of the invention is alsopresent/added in step (b).

The fermentation product, such as especially ethanol, may optionally berecovered after fermentation, e.g., by distillation. Suitablestarch-containing starting materials are listed in the section“Starch-containing materials”-section below. Contemplated enzymes arelisted in the “Enzymes”-section below. The liquefaction is preferablycarried out in the presence of an alpha-amylase. The fermentation ispreferably carried out in the presence of yeast, preferably a strain ofSaccharomyces. Suitable fermenting organisms are listed in the“Fermenting Organisms”-section below. In preferred embodiments step (b)and (c) are carried out sequentially or simultaneously (i.e., as SSFprocess).

In a particular embodiment, the process of the invention furthercomprises, prior to the step (a), the steps of:

x) reducing the particle size of the starch-containing material,preferably by milling; andy) forming a slurry comprising the starch-containing material and water.

The aqueous slurry may contain from 10-40 wt. %, preferably 25-35 wt. %starch-containing material. The slurry is heated to above thegelatinization temperature and alpha-amylase, preferably bacterialand/or acid fungal alpha-amylase, may be added to initiate liquefaction(thinning). The slurry may in an embodiment be jet-cooked to furthergelatinize the slurry before being subjected to an alpha-amylase in step(a) of the invention.

More specifically liquefaction may be carried out as a three-step hotslurry process. The slurry is heated to between 60-95° C., preferably80-85° C., and alpha-amylase is added to initiate liquefaction(thinning). Then the slurry may be jet-cooked at a temperature between95-140° C., preferably 105-125° C., for 1-15 minutes, preferably for3-10 minute, especially around 5 minutes. The slurry is cooled to 60-95°C. and more alpha-amylase is added to finalize hydrolysis (secondaryliquefaction). The liquefaction process is usually carried out at pH4.5-6.5, in particular at a pH between 5 and 6. Milled and liquefiedwhole grains are known as mash.

The saccharification in step (b) may be carried out using conditionswell know in the art. For instance, a full saccharification process maylast up to from about 24 to about 72 hours, however, it is common onlyto do a pre-saccharification of typically 40-90 minutes at a temperaturebetween 30-65° C., typically about 60° C., followed by completesaccharification during fermentation in a simultaneous saccharificationand fermentation process (SSF process). Saccharification is typicallycarried out at temperatures from 30-65° C., typically around 60° C., andat a pH between 4 and 5, normally at about pH 4.5.

The most widely used process in fermentation product, especiallyethanol, production is the simultaneous saccharification andfermentation (SSF) process, in which there is no holding stage for thesaccharification, meaning that fermenting organism, such as yeast, andenzyme(s) may be added together. SSF may typically be carried out at atemperature between 25° C. and 40° C., such as between 29° C. and 35°C., such as between 30° C. and 34° C., such as around 32° C. Accordingto the invention the temperature may be adjusted up or down duringfermentation.

In accordance with the present invention the fermentation step (c)includes, without limitation, fermentation processes used to producealcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citricacid, acetic acid, itaconic acid, lactic acid, gluconic acid); ketones(e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H₂ andCO₂); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins(e.g., riboflavin, B12, beta-carotene); and hormones. Preferredfermentation processes include alcohol fermentation processes, as arewell known in the art. Preferred fermentation processes are anaerobicfermentation processes, as are well known in the art.

Processes for Producing Fermentation Products from Un-GelatinizedStarch-Containinq

In this aspect the invention relates to processes for producing afermentation product from starch-containing material withoutgelatinization of the starch-containing material (i.e., uncookedstarch-containing material). According to the invention the desiredfermentation product, such as ethanol, can be produced withoutliquefying the aqueous slurry containing the starch-containing material.In one embodiment a process of the invention includes saccharifying(milled) starch-containing material, e.g., granular starch, below thegelatinization temperature in the presence of an alpha amylase toproduce sugars that can be fermented into the desired fermentationproduct by a suitable fermenting organism. In another embodiment aglucoamylase of the invention and an alpha amylase are used duringsaccharification and fermentation. In one aspect the invention relatesto a process for producing a fermentation product from starch-containingmaterial comprising:

(a) saccharifying starch-containing material with a mature glucoamylaseaccording to the invention, preferably having the sequence shown asamino acids 22 to 616 in SEQ ID NO: 2, at a temperature below theinitial gelatinization temperature of said starch-containing material,(b) fermenting using a fermenting organism.Steps (a) and (b) of the process of the invention may be carried outsequentially or simultaneously. In an embodiment, a slurry comprisingwater and starch-containing material, is prepared before step (a).

In a preferred embodiment step (a) includes addition of an alphaamylase.

The fermentation process may be carried out for a period of 1 to 250hours, preferably is from 25 to 190 hours, more preferably from 30 to180 hours, more preferably from 40 to 170 hours, even more preferablyfrom 50 to 160 hours, yet more preferably from 60 to 150 hours, even yetmore preferably from 70 to 140 hours, and most preferably from 80 to 130hours.

The term “initial gelatinization temperature” means the lowesttemperature at which gelatinization of the starch commences. Starchheated in water begins to gelatinize between 50° C. and 75° C.; theexact temperature of gelatinization depends on the specific starch, andcan readily be determined by the skilled artisan. Thus, the initialgelatinization temperature may vary according to the plant species, tothe particular variety of the plant species as well as with the growthconditions. In the context of this invention the initial gelatinizationtemperature of a given starch-containing material is the temperature atwhich birefringence is lost in 5% of the starch granules using themethod described by Gorinstein and Lii, 1192, Starch/Stärke 44(12):461-466.

Before step (a) a slurry of starch-containing material, such as granularstarch, having 10-55 wt. % dry solids, preferably 25-40 wt. % drysolids, more preferably 30-35 wt. % dry solids of starch-containingmaterial may be prepared. The slurry may include water and/or processwaters, such as stillage (backset), scrubber water, evaporatorcondensate or distillate, side stripper water from distillation, orother fermentation product plant process water. Because the process ofthe invention is carried out below the gelatinization temperature andthus no significant viscosity increase takes place, high levels ofstillage may be used if desired. In an embodiment the aqueous slurrycontains from about 1 to about 70 vol. % stillage, preferably 15-60%vol. % stillage, especially from about 30 to 50 vol. % stillage.

The starch-containing material may be prepared by reducing the particlesize, preferably by dry or wet milling, to 0.05 to 3.0 mm, preferably0.1-0.5 mm. After being subjected to a process of the invention at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or preferably at least99% of the dry solids of the starch-containing material is convertedinto a soluble starch hydrolysate.

The process of the invention is conducted at a temperature below theinitial gelatinization temperature. Preferably the temperature at whichstep (a) is carried out is between 30-75° C., preferably between 45-60°C.

In a preferred embodiment step (a) and step (b) are carried out as asequential or simultaneous saccharification and fermentation process. Insuch preferred embodiment the process is typically carried at atemperature between 25° C. and 40° C., such as between 29° C. and 35°C., such as between 30° C. and 34° C., such as around 32° C. Accordingto the invention the temperature may be adjusted up or down duringfermentation.

In an embodiment simultaneous saccharification and fermentation iscarried out so that the sugar level, such as glucose level, is kept at alow level such as below 6 wt. %, preferably below about 3 wt. %,preferably below about 2 wt. %, more preferred below about 1 wt. %.,even more preferred below about 0.5 wt. %, or even more preferred 0.25wt. %, such as below about 0.1 wt. %. Such low levels of sugar can beaccomplished by simply employing adjusted quantities of enzyme andfermenting organism. A skilled person in the art can easily determinewhich quantities of enzyme and fermenting organism to use. The employedquantities of enzyme and fermenting organism may also be selected tomaintain low concentrations of maltose in the fermentation broth. Forinstance, the maltose level may be kept below about 0.5 wt. % or belowabout 0.2 wt. %.

The process may be carried out at a pH in the range between 3 and 7,preferably from pH 3.5 to 6, or more preferably from pH 4 to 5.

The glucoamylase of the present invention is highly thermostable, so thepre-saccharification and/or saccharification of the present inventioncan be carried at a higher temperature than the conventionalpre-saccharification and/or saccharification. In one embodiment aprocess of the invention includes pre-saccharifying starch-containingmaterial before simultaneous saccharification and fermentation (SSF)process. The pre-saccharification can be carried out at a hightemperature (for example, 50-85° C., preferably 60-75° C.) before movinginto SSF.

Starch-Containing Materials

Any suitable starch-containing starting material, including granularstarch, may be used according to the present invention. The startingmaterial is generally selected based on the desired fermentationproduct. Examples of starch-containing starting materials, suitable foruse in a process of present invention, include tubers, roots, stems,whole grains, corns, cobs, wheat, barley, rye, milo, sago, cassava,tapioca, sorghum, rice peas, beans, or sweet potatoes, or mixturesthereof, or cereals, sugar-containing raw materials, such as molasses,fruit materials, sugar cane or sugar beet, potatoes, andcellulose-containing materials, such as wood or plant residues, ormixtures thereof. Contemplated are both waxy and non-waxy types of cornand barley.

Fermenting Organisms

“Fermenting organism” refers to any organism, including bacterial andfungal organisms, suitable for use in a fermentation process and capableof producing desired a fermentation product. Especially suitablefermenting organisms are able to ferment, i.e., convert, sugars, such asglucose or maltose, directly or indirectly into the desired fermentationproduct. Examples of fermenting organisms include fungal organisms, suchas yeast. Preferred yeast includes strains of Saccharomyces spp., inparticular, Saccharomyces cerevisiae. Commercially available yeastinclude, e.g., Red Star™/Lesaffre Ethanol Red (available from RedStar/Lesaffre, USA) FALI (available from Fleischmann's Yeast, a divisionof Burns Philp Food Inc., USA), SUPERSTART (available from Alltech),GERT STRAND (available from Gert Strand AB, Sweden) and FERMIOL(available from DSM Specialties).

Enzymes Glucoamylase

The glucoamylase is preferably a glucoamylase of the invention. However,as mentioned above a glucoamylase of the invention may also be combinedwith other glucoamylases.

The glucoamylase may added in an amount of 0.001 to 10 AGU/g DS,preferably from 0.01 to 5 AGU/g DS, such as around 0.05, 0.1, 0.3, 0.5,1 or 2 AGU/g DS, especially 0.05 to 0.5 AGU/g DS; or 0.02-20 AGU/g DS,preferably 0.1-10 AGU/g DS.

Alpha-Amylase

The alpha-amylase may according to the invention be of any origin.Preferred are alpha-amylases of fungal or bacterial origin.

In a preferred aspect the alpha-amylase is an acid alpha-amylase, e.g.,fungal acid alpha-amylase or bacterial acid alpha-amylase. The term“acid alpha-amylase” means an alpha-amylase (E.C. 3.2.1.1) which addedin an effective amount has activity optimum at a pH in the range of 3 to7, preferably from 3.5 to 6, or more preferably from 4-5.

Bacterial Alpha-Amylases

According to the invention a bacterial alpha-amylase may preferably bederived from the genus Bacillus.

In a preferred aspect the Bacillus alpha-amylase is derived from astrain of B. licheniformis, B. amyloliquefaciens, B. subtilis or B.stearothermophilus, but may also be derived from other Bacillus sp.Specific examples of contemplated alpha-amylases include the Bacilluslicheniformis alpha-amylase (BLA) shown in SEQ ID NO: 4 in WO 99/19467,the Bacillus amyloliquefaciens alpha-amylase (BAN) shown in SEQ ID NO: 5in WO 99/19467, and the Bacillus stearothermophilus alpha-amylase (BSG)shown in SEQ ID NO: 3 in WO 99/19467. In an embodiment of the inventionthe alpha-amylase is an enzyme having a degree of identity of at least60%, preferably at least 70%, more preferred at least 80%, even morepreferred at least 90%, such as at least 95%, at least 96%, at least97%, at least 98% or at least 99% identity to any of the sequences shownas SEQ ID NOS: 1, 2, 3, 4, or 5, respectively, in WO 99/19467.

The Bacillus alpha-amylase may also be a variant and/or hybrid,especially one described in any of WO 96/23873, WO 96/23874, WO97/41213, WO 99/19467, WO 00/60059, and WO 02/10355 (all documentshereby incorporated by reference). Specifically contemplatedalpha-amylase variants are disclosed in U.S. Pat. Nos. 6,093,562,6,297,038 or U.S. Pat. No. 6,187,576 (hereby incorporated by reference)and include Bacillus stearothermophilus alpha-amylase (BSGalpha-amylase) variants having a deletion of one or two amino acid inposition 179 to 182, preferably a double deletion disclosed in WO1196/023873—see e.g., page 20, lines 1-10 (hereby incorporated byreference), preferably corresponding to delta (181-182) compared to thewild-type BSG alpha-amylase amino acid sequence set forth in SEQ ID NO:3 disclosed in WO 99/19467 or deletion of amino acids 179 and 180 usingSEQ ID NO: 3 in WO 99/19467 for numbering (which reference is herebyincorporated by reference). Even more preferred are Bacillusalpha-amylases, especially Bacillus stearothermophilus alpha-amylase,which have a double deletion corresponding to delta (181-182) andfurther comprise a N193F substitution (also denoted 1181*+G182*+N193F)compared to the wild-type BSG alpha-amylase amino acid sequence setforth in SEQ ID NO: 3 disclosed in WO 99/19467.

The alpha-amylase may also be a maltogenic alpha-amylase. A “maltogenicalpha-amylase” (glucan 1,4-alpha-maltohydrolase, E.C. 3.2.1.133) is ableto hydrolyze amylose and amylopectin to maltose in thealpha-configuration. A maltogenic alpha-amylase from Bacillusstearothermophilus strain NCIB 11837 is commercially available fromNovozymes NS, Denmark. The maltogenic alpha-amylase is described in U.S.Pat. Nos. 4,598,048, 4,604,355 and 6,162,628, which are herebyincorporated by reference.

Bacterial Hybrid Alpha-Amylases

A hybrid alpha-amylase specifically contemplated comprises 445C-terminal amino acid residues of the Bacillus licheniformisalpha-amylase (shown as SEQ ID NO: 4 in WO 99/19467) and the 37N-terminal amino acid residues of the alpha-amylase derived fromBacillus amyloliquefaciens (shown as SEQ ID NO: 3 in WO 99/194676), withone or more, especially all, of the following substitutions:

G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S (using the Bacilluslicheniformis numbering). Also preferred are variants having one or moreof the following mutations (or corresponding mutations in other Bacillusalpha-amylase backbones): H154Y, A181T, N190F, A209V and Q264S and/ordeletion of two residues between positions 176 and 179, preferablydeletion of E178 and G179 (using the SEQ ID NO: 5 numbering of WO99/19467).

Fungal Alpha-Amylases

Fungal acid alpha-amylases include acid alpha-amylases derived from astrain of the genus Aspergillus, such as Aspergillus oryzae, Aspergillusniger, or Aspergillus kawachii alpha-amylases.

A preferred acid fungal alpha-amylase is a Fungamyl-like alpha-amylasewhich is preferably derived from a strain of Aspergillus oryzae. In thepresent disclosure, the term “Fungamyl-like alpha-amylase” indicates analpha-amylase which exhibits a high identity, i.e. more than 70%, morethan 75%, more than 80%, more than 85% more than 90%, more than 95%,more than 96%, more than 97%, more than 98%, more than 99% or even 100%identity to the mature part of the amino acid sequence shown in SEQ IDNO: 10 in WO 96/23874.

Another preferred acid alpha-amylase is derived from a strainAspergillus niger. In a preferred aspect the acid fungal alpha-amylaseis the one from A. niger disclosed as “AMYA_ASPNG” in theSwiss-prot/TeEMBL database under the primary accession no. P56271 anddescribed in more detail in WO 89/01969 (Example 3). The acidAspergillus niger acid alpha-amylase is also shown as SEQ ID NO: 1 in WO2004/080923 (Novozymes) which is hereby incorporated by reference. Alsovariants of said acid fungal amylase having at least 70% identity, suchas at least 80% or even at least 90% identity, such as at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identity to SEQID NO: 1 in WO 2004/080923 are contemplated.

In a preferred aspect the alpha-amylase is derived from Aspergilluskawachii and disclosed by Kaneko et al. J. Ferment. Bioeng.81:292-298(1196) “Molecular-cloning and determination of thenucleotide-sequence of a gene encoding an acid-stable alpha-amylase fromAspergillus kawachii”; and further as EMBL:#AB008370.

The fungal acid alpha-amylase may also be a wild-type enzyme comprisinga carbohydrate-binding module (CBM) and an alpha-amylase catalyticdomain (i.e., a none-hybrid), or a variant thereof. In an embodiment thewild-type acid alpha-amylase is derived from a strain of Aspergilluskawachii.

Fungal Hybrid Alpha-Amylases

In a preferred aspect the fungal acid alpha-amylase is a hybridalpha-amylase. Preferred examples of fungal hybrid alpha-amylasesinclude the ones disclosed in WO 2005/003311 or U.S. Patent Publicationno. 2005/0054071 (Novozymes) or US patent application No. 2006/0148054(Novozymes) which is hereby incorporated by reference. A hybridalpha-amylase may comprise an alpha-amylase catalytic domain (CD) and acarbohydrate-binding domain/module (CBM) and optional a linker.

Specific examples of contemplated hybrid alpha-amylases include, but notlimited to those disclosed in U.S. patent application No. 2006/0148054including Fungamyl variant with catalytic domain JA118 and Atheliarolfsii SBD (SEQ ID NO: 100 in U.S. application No. 2006/0148054),Rhizomucor pusillus alpha-amylase with Athelia rolfsii AMG linker andSBD (SEQ ID NO: 101 in U.S. application No. 2006/0148054) and Meripilusgiganteus alpha-amylase with Athelia rolfsii glucoamylase linker and SBD(SEQ ID NO: 102 in U.S. application No. 2006/0148054); and Rhizomucorpusillus alpha-amylase with Aspergillus niger glucoamylase linker andCBM (SEQ ID NO 2 in international publication No. WO2007/144424).

Other specific examples of contemplated hybrid alpha-amylases include,but not limited to those disclosed in U.S. Patent Publication no.2005/0054071, including those disclosed in Table 3 on page 15, such asAspergillus niger alpha-amylase with Aspergillus kawachii linker andstarch binding domain.

Commercial Alpha-Amylase Products

Preferred commercial compositions comprising alpha-amylase includeMYCOLASE from DSM (Gist Brocades), BAN™, TERMAMYL™ SC, FUNGAMYL™,LIQUOZYME™ SC, LIQUOZYME™ SC DS, and SAN™ SUPER, SAN™ EXTRA L (NovozymesNS) and CLARASE™ L-40,000, DEX-LO™, SPEZYME™ FRED, SPEZYME™ AA, SPEZYME™Ethyl, and SPEZYME™ DELTA AA (Genencor Int.)

The present invention is further described by the following numberedparagraphs:

Paragraph[1]. A glucoamylase variant comprising a substitution at one ormore positions corresponding to positions 45, 46, 61, 86, 119, 120, 318,348 of the polypeptide of SEQ ID NO: 3 wherein the variant hasglucoamylase activity and wherein the variant has at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%, but less than 100% sequence identityto the polypeptide of SEQ ID NO: 3.

Paragraph[2]. The variant of paragraph 1, which is a variant of a parentglucoamylase selected from the group consisting of: a polypeptide havingat least 75% sequence identity to the mature polypeptide of SEQ ID NO:2; a polypeptide encoded by a polynucleotide having at least 75%identity to the mature polypeptide coding sequence of SEQ ID NO: 1; anda fragment of the polypeptide of SEQ ID NO: 3, which has glucoamylaseactivity.

Paragraph[3]. The variant of paragraph 2, wherein the parentglucoamylase has at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to the polypeptide of SEQ ID NO: 3.

Paragraph[4]. The variant of any of paragraphs 2-3, wherein the parentglucoamylase is encoded by a polynucleotide having at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to themature polypeptide coding sequence of SEQ ID NO: 1.

Paragraph[5]. The variant of any of paragraphs 2-4, wherein the parentglucoamylase comprises or consists of the polypeptide of SEQ ID NO: 3.

Paragraph[6]. The variant of any of paragraphs 2-5, wherein the parentglucoamylase is a fragment of the mature polypeptide of SEQ ID NO: 3,wherein the fragment has glucoamylase activity.

Paragraph[7]. The variant of any of paragraphs 1-6, wherein the maturepolypeptide of SEQ ID NO: 2 is SEQ ID NO: 3.

Paragraph[8]. The variant of any of paragraphs 1-7, which has at least75%, at least 80%, at least 85%, at least 90%, at least 95% identity, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100%, sequence identity to the amino acid sequence of the parentglucoamylase.

Paragraph[9]. The variant of any of paragraphs 1-8, which has at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%sequence identity to the polypeptide of SEQ ID NO: 3.

Paragraph[10]. The variant of any of paragraphs 1-9, wherein the numberof substitutions is 1-20, e.g., 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 substitutions.

Paragraph[11]. The variant of any of paragraphs 1-10, which comprises asubstitution at a position corresponding to position 45.

Paragraph[12]. The variant of paragraph 11, wherein the substitution iswith Lysine.

Paragraph[13]. The variant of any of paragraphs 1-10, which comprises asubstitution at a position corresponding to position 46.

Paragraph[14]. The variant of paragraph 13, wherein the substitution iswith Histidine.

Paragraph[15]. The variant of any of paragraphs 1-10, which comprises asubstitution at a position corresponding to position 61.

Paragraph[16]. The variant of paragraph 15, wherein the substitution iswith Threonine or Alanine.

Paragraph[17]. The variant of any of paragraphs 1-10, which comprises asubstitution at a position corresponding to position 86.

Paragraph[18]. The variant of paragraph 17, wherein the substitution iswith Cysteine.

Paragraph[19]. The variant of any of paragraphs 1-10, which comprises asubstitution at a position corresponding to position 119.

Paragraph[20]. The variant of paragraph 19, wherein the substitution iswith Tryptophan.

Paragraph[21]. The variant of any of paragraphs 1-10, which comprises asubstitution at a position corresponding to position 120.

Paragraph[22]. The variant of paragraph 21, wherein the substitution iswith Glycine.

Paragraph[23]. The variant of any of paragraphs 1-10, which comprises asubstitution at a position corresponding to position 318.

Paragraph[24]. The variant of paragraph 23, wherein the substitution iswith Tryptophan.

Paragraph[25]. The variant of any of paragraphs 1-10, which comprises asubstitution at a position corresponding to position 348.

Paragraph[26]. The variant of paragraph 25, wherein the substitution iswith Threonine.

Paragraph[27]. The variant of any of paragraphs 1-26, which comprises asubstitution at two positions corresponding to any of positions 45, 46,61, 86, 119, 120, 318 and 348.

Paragraph[28]. The variant of any of paragraphs 1-27, which comprises asubstitution at three positions corresponding to any of positions 45,46, 61, 86, 119, 120, 318 and 348.

Paragraph[29]. The variant of any of paragraphs 1-28, which comprises asubstitution at four positions corresponding to any of positions 59, 95,119, 121, 18, 426, and 316.

Paragraph[30]. The variant of any of paragraphs 1-29, which comprises asubstitution at five positions corresponding to any of positions 45, 46,61, 86, 119, 120, 318 and 348.

Paragraph[31]. The variant of any of paragraphs 1-30, which comprises asubstitution at six positions corresponding to any of positions 45, 46,61, 86, 119, 120, 318 and 348.

Paragraph[32]. The variant of any of paragraphs 1-31, which comprises asubstitution at each position corresponding to positions 45, 46, 61, 86,119, 120, 318 and 348.

Paragraph[33]. The variant according to paragraphs 1-32, wherein thevariant comprises or consists of one or more substitutions selected fromthe group consisting of T45K, S46H, V61A, V61T, S86C, T119W, D120G,M318W, N348T and wherein the variant has glucoamylase activity andwherein the variant has at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

Paragraph[34]. The variant of any of the preceding paragraphs, whereinthe variant comprises at least one of the following substitutions orcombinations of substitutions:

T119W; V61A; D120G; S86C+T119W; T45K+T119W; T119W+D120G; T45K+D120G;V61T+T119W; D120G+M318W; T45K+S46H; T119W+D120G+N348T;T119W+D120G+M318W; S86C+T119W+D120G; S46H+T119W+D120G;

T45K+T119W+D120G and wherein the variant has glucoamylase activity andwherein the variant has at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

Paragraph[35]. The variant according to paragraphs 1-34, which has animproved property relative to the parent, wherein the improved propertyis increased thermostability.

Paragraph[36]. A composition comprising the polypeptide of any ofparagraphs 1-35.

Paragraph[37]. The composition according to paragraph 36, comprising analpha-amylase and a polypeptide of any of paragraphs 1-35.

Paragraph[38]. The composition according to paragraph 36, comprising analpha-amylase, an isoamylase and a polypeptide of any of claims 1-35.

Paragraph[39]. The composition according to paragraph 36, comprising anpullulanase and a polypeptide of any of paragraphs 1-35.

Paragraph[40]. A use of a polypeptide of any of paragraphs 1-35 forproduction of syrup and/or a fermentation product.

Paragraph[41]. The use according to paragraph 40, wherein the startingmaterial is gelatinized or un-gelatinized starch-containing material.

Paragraph[42]. A process of producing a fermentation product fromstarch-containing material comprising the steps of: (a) liquefyingstarch-containing material in the presence of an alpha amylase; (b)saccharifying the liquefied material; and (c) fermenting with afermenting organism; wherein step (a) and/or step (b) is carried outusing at least a glucoamylase variant of any of paragraphs 1-35.

Paragraph[43]. A process of producing a fermentation product fromstarch-containing material, comprising the steps of: (a) saccharifyingstarch-containing material at a temperature below the initialgelatinization temperature of said starch-containing material; and (b)fermenting with a fermenting organism, wherein step (a) is carried outusing at least a glucoamylase variant of any of paragraphs 1-35.

Paragraph[44] A process of producing a syrup product fromstarch-containing material, comprising the step of: saccharifyingstarch-containing material at a temperature below the initialgelatinization temperature of said starch-containing material in thepresence of a glucoamylase variant of any of paragraphs 1-40.

Paragraph[45]. A process of producing a syrup product fromstarch-containing material, comprising the step of: (a) liquefyingstarch-containing material in the presence of an alpha amylase; (b)saccharifying the liquefied material in the presence of a glucoamylasevariant of any of paragraphs 1-40.

Paragraph[46] The process according to paragraph 45, wherein step b)further comprises adding a pullulanase.

Paragraph[47]. The process according to paragraphs 45 and 46, whereinthe saccharification temperature is the range from 40 to 65° C., moreparticular from 50 to 62° C., more particular from 59 to 62° C.

Paragraph[48] An isolated polynucleotide encoding the variant of any ofparagraphs 1-35.

Paragraph[49]. A nucleic acid construct comprising the polynucleotide ofparagraph 42.

Paragraph[50]. An expression vector comprising the polynucleotide ofparagraph 42.

Paragraph[51]. A host cell comprising the polynucleotide of paragraph42.

Paragraph[52]. A method of producing a glucoamylase variant, comprising:cultivating the host cell of paragraph 51 under conditions suitable forexpression of the variant; and recovering the variant glucoamylase.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Materials and Methods Glucoamylase Activity

Glucoamylase activity may be measured in AGU Units.

Glucoamylase Activity (AGU)

The Glucoamylase Unit (AGU) is defined as the amount of enzyme, whichhydrolyses 1 micromole maltose per minute under the standard conditions(37° C., pH 4.3, substrate: maltose 100 mM, buffer: acetate 0.1 M,reaction time 6 minutes as set out in the glucoamylase incubationbelow), thereby generating glucose.

glucoamylase incubation: Substrate: maltose 100 mM Buffer: acetate 0.1MpH: 4.30 ± 0.05 Incubation temperature: 37° C. ± 1 Reaction time: 6minutes Enzyme working range: 0.5-4.0 AGU/mL

The analysis principle is described by 3 reaction steps:

Step 1 is an enzyme reaction:

Glucoamylase (AMG), EC 3.2.1.3 (exo-alpha-1,4-glucan-glucohydrolase),hydrolyzes maltose to form alpha-D-glucose. After incubation, thereaction is stopped with NaOH.

Steps 2 and 3 result in an endpoint reaction:

Glucose is phosphorylated by ATP, in a reaction catalyzed by hexokinase.The glucose-6-phosphate formed is oxidized to 6-phosphogluconate byglucose-6-phosphate dehydrogenase. In this same reaction, an equimolaramount of NAD+ is reduced to NADH with a resulting increase inabsorbance at 340 nm. An autoanalyzer system such as Konelab 30 Analyzer(Thermo Fisher Scientific) may be used.

Color reaction Tris approx. 35 mM ATP 0.7 mM NAD⁺ 0.7 mM Mg²⁺ 1.8 mMHexokinase >850 U/L Glucose-6-P-DH >850 U/L pH approx. 7.8 Temperature37.0° C. ± 1.0 ° C. Reaction time 420 sec Wavelength 340 nmPurification of site-directed Tc AMG variants

The protein purification was carried out by affinity chromatographyusing α-cyclodextrin. A-cyclodextrin acts as a substrate analogue forTc-AMG (Trametes cingulata AMG), the enzyme does not degrade thecompound and can be eluted using α-cyclodextrin in the elution buffer.

Preparation of purification Matrix

Materials

-   -   Alfa-cyclodextrin (Fluka 28705)    -   DVs agarose activated with di-vinyl sulfone (Mini leak high        Kem-en-Tec 1013F)    -   Coupling buffer; K2HPO4 (0.5M, pH 11)    -   Wash buffer; Phosphate buffer (0.5M, pH 7)    -   Blocking buffer; Ethanolamine (0.1 M) (E9508)

Procedure

Wash the matrix (50 ml) with 5×100 ml milliQ water, on suction filter(sintered glass, G4). α-cyclodextrin (6 g) was dissolved in couplingbuffer (100 ml). The suck dried matrix was transferred to theα-cyclodextrin solution and rotated overnight. The coupled matrix waswashed with wash buffer (5×100 ml). The matrix was blocked with blockingbuffer for 2 hours with rotation. The matrix was washed with ultra purewater (5×100 ml), on suction filter (sintered glass, G4). The coupledmatrix was stored in ethanol (20%) for future use.

Filtration

The Aspergillus fermentation was filtered first through a sandwich ofglass fibre filters (GF/A, C, F, in the order from the top) and thensterile filtered through 0.2μ filter using a tangential flow filtercartridge fixed to GE QuixStand.

Purification

-   -   Matrix: Alfa-cyclodextrin (20 ml)    -   Equilibration buffer (A buffer): 50 mM Na-Acetate pH 4,5    -   B buffer: 50 mM Na-Acetate, 1% alfa-CD (fluka28705) pH 4,5    -   Elution: isocratic in elution buffer (5% B and 95% A)    -   Flow rate 8 ml/min    -   Fraction collection size 14 ml    -   The matrix was packed in a glass column and equilibrated with        the equilibration buffer (200 ml)    -   The filtered fermentation broth (sample) was passed through the        column    -   The post sample wash was performed by passing equilibration        buffer (200 ml).    -   The protein was eluted by passing elution buffer (200 ml)    -   The fractions (14 ml) which had absorbance at 280 was pooled.    -   This pool was designated as the purified sample.

Glucoamylase Stability Assay—Residual Activity (Cultured Supernatant)Reagents:

1. Culture supernatant sample: 10× dilution were made for the culturesupernatant using 100 mM Acetate buffer pH 4.5

2. Stock Solution of 1M Acetate Buffer pH 4.5 (500 ml)

a. Sodium acetate (Sigma S7546 Batch No. 125K0085): (41.015 g)b. pH adjusted with acetic acidc. Volume made up to 500 ml using Type I water

Stored at 4° C.

3. 0.1M Borax (Sodium Tetraborate) solution (500 ml)a. Di-Sodium tetraborate decahydrate: (19.068 g)b. Dissolved in Type I water and volume made up to 500 mlStored at Room temperature4. p-Nitrophenyl-α-D-glucopyranoside (pNPG) Substrate: 0.2% (6.66 mM)a. 200 mg of pNPG

b. 10 ml of 1M Acetate Buffer pH 4.5

c. Type I water up to 100 mlStored at 4° C. after preparation till use (should be prepared freshlyevery day for assay)

5. Standard Solution

a. Tc-AMG WT Purified sample is used as standard in the assayb. Concentration of the enzyme is 0, 2, 4, 6, 8, 12, 16 and 20 μg/ml wasprepared and 90 μl of this is taken in the assay

Procedure

90 μl of diluted culture supernatant was taken in PCR plates intriplicates and standard solution is pipetted into the plate with oneplate will be control. One set was kept at 4° C. as control. Second setwas subjected to stress at 60° C. for 20 min one plate at a time usingThermal cycler. Immediately after stress cooled to 4° C. and stored inrefrigerator till assay. To the stressed plate 90 ul of standardsolution is pipette and stored at 4° C. till the time of assay.

Enzyme Assay

One PCR Plate either 4° C. control or stressed sample are taken whichcontains 90 μl of samples and kept in the thermal cycler which is at 4°C. 30 μl of 0.2% pNPG substrate solution is added to the enzyme andincubated at 40° C. for 30 min. Reaction is stopped using 120 μl of 0.1MSodium tetraborate solution. Similarly the second set for control, 2plates of stressed sample is assayed as mentioned above and followed bystressed samples in the same way. From this 120 μl of the sample istransferred to 96 Well MTP Plate and absorbance measured at 405 nm.

Glucoamylase Stability Assay—Residual Activity (Purified Sample)Reagents:

1. Purified protein sample:—Normalized to 10 μg/ml

2. Stock Solution of 1M Acetate Buffer pH 4.5 (500 ml)

Sodium acetate (Sigma S7546 Batch No. 125K0085): (41.015 g)pH adjusted with acetic acidVolume made up to 500 ml using Type I water

Stored at 4° C.

3. 0.1 M Borax (Sodium Tetraborate) solution (500 ml)Di-Sodium tetraborate decahydrate: (19.068 g)Dissolved in Type I water and volume made up to 500 ml

Stored at Room Temperature

4. pNPG Substrate: 0.2% (6.66 mM)200 mg of pNPG

10 ml of 1M Acetate Buffer pH 4.5

Type 1 water up to 100 mlStored at 4° C. after preparation till use (should be prepared freshlyevery day for assay)5. Standard solutionTcAMG WT Purified sample is used as standard in the assayConcentration of the enzyme is 0, 2, 4, 6, 8, 12, 16 and 20 μg/ml wasprepared and 90 μl of this is taken in the assay

Procedure

90 μl of diluted enzyme sample is taken in PCR plates in triplicates,and standard solution is also pipette into the plate for one plate whichwill be control. One set was kept at 4° C. as control. Second set wassubjected to stress at 60° C. for 20 min one plate at a time usingThermal cycler. Immediately after stress cooled to 4° C. and stored inrefrigerator till assay. To the stressed plate 90 μl of standardsolution is pipette and stored at 4° C. till the time of assay.

Enzyme Assay

One PCR Plate either 4° C. control or stressed sample are taken whichcontains 90 μl of samples and kept in the thermal cycler at 4° C. 30 μlof 0.2% pNPG substrate solution is added to the enzyme and incubated at40° C. for 30 min. Reaction is stopped using 120 μl of 0.1M Sodiumtetraborate solution. Similarly the second set which contains plates ofstressed sample is assayed as mentioned above and followed by stressedsamples in the same way. From this 120 μl of the sample is transferredto 96 Well MTP Plate and absorbance measured at 405 nm.

Thermostability Analysis by Differential Scanning Calorimitry (DSC)

The improved variants selected from the residual activity assay werealso tested using Differential Scanning calorimitry as described byFreire, E. (1995) (Methods. Mol. Biol. 41, 191-218).

The thermo-stability of the purified Tc-AMG derived variants weredetermined at pH 4.5 (50 mM Sodium Acetate) by Differential Scanningcalorimetry (DSC) using a VP-Capillary Differential Scanning calorimeter(MicroCal Inc., Piscataway, N.J., USA). The thermal denaturationtemperature, Td (° C.), was taken as the top of the denaturation peak(major endothermic peak) in thermograms (Cp vs. T) obtained afterheating enzyme solutions in selected buffers (50 mM Sodium Acetate, pH4.5) at a constant programmed heating rate of 200 K/hr.

Sample- and reference-solutions (approximately 0.3 ml of 0.4 mg/ml) wereloaded into the calorimeter (reference: buffer without enzyme) fromstorage conditions at 10° C. and thermally pre-equilibrated for 10minutes at 20° C. prior to DSC scan from 20° C. to 110° C. Denaturationtemperatures were determined with an accuracy of approximately +/−1° C.

Example 1 Cloning and Expression of the Glucoamylase (AMG) Gene fromTrametes cingulate

An expression construct, pHUda440, containing the wild type T. cingulataAMG was described in WO2006/069289 in Example 4. This constructcomprises the polynucleotide sequence shown herein as SEQ ID NO: 1 andencoding the mature polypeptide shown herein as amino acids 18 to 576 ofSEQ ID NO: 2 corresponding to SEQ ID NO: 3. WO2006/069289 furtherdescribes the expression of wild type T. cingulata AMG from pHUda440.

Example 2 Variant Generation by Site-Directed Mutagenesis

Based on the plasmid disclosed in WO2006/069289 expressing the wild typeT. cingulata AMG, each of the specific site directed mutations asdisclosed herein can be generated by designing the appropriate primers.The resulting genes can be cloned and expressed by methods known to theskilled person.

The specific variants constructed and tested for improvedthermo-stability disclosed in the below table.

Example 3 Improved Thermo-Stability of Specific Single SubstitutionsMeasured by DSC and Residual Glucoamylase Activity

Primary screening was carried out using culture supernatant samples andmeasuring residual activity using the glucoamylase thermal stabilityassay described above. This initial analysis identified improvedthermo-stable variants compared to the wild type. The improved variantswere selected based on the assay results. The Assay reaction was carriedout in 100 mM Sodium acetate buffer pH 4.5. The reaction mixtureconsists of 90ul of 10 times (Buffer 100 mM Acetate buffer pH 4.5)diluted culture supernatant and stressed at 60° C. for 20 min and to thesame sample 30 μl of the substrate (0.2% in buffer). Samples wereincubated at 40° C. for 30 min and the reaction was stopped by adding120 μl of sodium tetraborate and the absorbance measured at 405 nm.Similarly control reactions were done which had not been subjected tothermal stress. The control activity was considered as 100% and theresidual activity was calculated relative to the control. The selectedvariants should have improved thermo-stability over the wt glucoamylase.The improved variants are listed below in the table.

TABLE 1 Residual activity and DSC data of single substituted variantsMutation % Residual Activity Td (Deg C) T45K 67 77.2 V61A 68 74.6 S46H75 76.6 V61T 78 75.9 S86C 86 75.4 T119W 80 76.7 D120G 86 78.5 M318W 7976.0 N348T 80 75.3 Wild Type 65 75.3

Example 4 Improved Thermo-Stability of Specific Combinations ofSubstitutions Measured by DSC and Residual Glucoamylase Activity

Some of improved variants were chosen for making specific combinationsof double or triple substitutions. The specific combinations were testedby measuring residual glucoamylase activity and thermal meltingtemperature by DSC.

The improved variants are listed below in the table 2

TABLE 2 Residual activity and DSC of combination of substituted variantsMutation % Residual Activity Td (Deg C) S86C + T119W 75 77.3 T45K +T119W 84 78.3 T119W + D120G 96 79.6 T45K + D120G 94 79.0 V61T + T119W 7477.3 D120G + M318W 84 77.7 T45K + S46H 77 77.0 T119W + D120G + N348T 8278.0 T119W + D120G + M318W 85 79.4 S46H + T119W + D120G 86 79.6 T45K +T119W + D120G 60 80.4 Wild Type 60 75.2

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1. A glucoamylase variant comprising a substitution at one or morepositions corresponding to positions 45, 46, 61, 86, 119, 120, 318, 348of the polypeptide of SEQ ID NO: 3 wherein the variant has glucoamylaseactivity and wherein the variant has at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100% sequence identity to thepolypeptide of SEQ ID NO:
 3. 2. The variant according to claim 1,wherein the number of substitutions is 1-20, e.g., 1-10 and 1-5, such as1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions.
 3. The variant of claim1, wherein the variant comprises or consists of one or moresubstitutions selected from the group consisting of T45K, S46H, V61A,V61T, S86C, T119W, D120G, M318W, N348T and wherein the variant hasglucoamylase activity and wherein the variant has at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%, but less than 100% sequence identityto the polypeptide of SEQ ID NO:
 3. 4. The variant of claim 1, whereinthe variant comprises at least one of the following substitutions orcombinations of substitutions: T119W; V61A; D120G; S86C+T119W;T45K+T119W; T119W+D120G; T45K+D120G; V61T+T119W; D120G+M318W; T45K+S46H;T119W+D120G+N348T; T119W+D120G+M318W; S86C+T119W+D120G;S46H+T119W+D120G; T45K+T119W+D120G and wherein the variant hasglucoamylase activity and wherein the variant has at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%, but less than 100% sequence identityto the polypeptide of SEQ ID NO:
 3. 5. The variant of claim 1, which hasan improved property relative to the parent, wherein the improvedproperty is increased thermostability.
 6. A composition comprising thepolypeptide of claim
 1. 7. The composition according to claim 6,comprising an alpha-amylase and a polypeptide of claim
 1. 8. (canceled)9. A process of producing a fermentation product from starch-containingmaterial comprising the steps of: (a) liquefying starch-containingmaterial in the presence of an alpha amylase; (b) saccharifying theliquefied material; and (c) fermenting with a fermenting organism;wherein step (a) and/or step (b) is carried out using at least aglucoamylase variant of claim
 1. 10. A process of producing a syrupproduct from starch-containing material, comprising the step of: (a)liquefying starch-containing material in the presence of an alphaamylase; (b) saccharifying the liquefied material in the presence of avariant glucoamylase of claim
 1. 11. An isolated polynucleotide encodingthe variant of claim
 1. 12. A nucleic acid construct comprising thepolynucleotide of claim
 11. 13. An expression vector comprising thepolynucleotide of claim
 11. 14. A host cell comprising thepolynucleotide of claim
 11. 15. A method of producing a glucoamylasevariant, comprising: cultivating the host cell of claim 14 underconditions suitable for expression of the variant; and recovering thevariant glucoamylase.